Allele-specific activators and inhibitors for kinesin - PubMed (original) (raw)

Allele-specific activators and inhibitors for kinesin

T M Kapoor et al. Proc Natl Acad Sci U S A. 1999.

Abstract

Members of the kinesin superfamily are force-generating ATPases that drive movement and influence cytoskeleton organization in cells. Often, more than one kinesin is implicated in a cellular process, and many kinesins are proposed to have overlapping functions. By using conventional kinesin as a model system, we have developed an approach to activate or inhibit a specific kinesin allele in the presence of other similar motor proteins. Modified ATP analogs are described that do not activate either conventional kinesin or another superfamily member, Eg5. However, a kinesin allele with Arg-14 in its nucleotide binding pocket mutated to alanine can use a subset of these nucleotide analogs to drive microtubule gliding. Cyclopentyl-ATP is one such analog. Cyclopentyl-adenylylimidodiphosphate, a nonhydrolyzable form of this analog, inhibits the mutant allele in microtubule-gliding assays, but not wild-type kinesin or Eg5. We anticipate that the incorporation of kinesin mutants and allele-specific activators and inhibitors in in vitro assays should clarify the role of individual motor proteins in complex cellular processes.

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Figures

Figure 1

Figure 1

The nucleotide binding pocket of human conventional kinesin bound to ADP. Side chains of residues within 6 Å of the N6 nitrogen of the nucleotide are highlighted. The figure was created with

ribbons

(41).

Figure 2

Figure 2

(A) Synthesis of the ATP and AMPPNP analogs. (B) Structures of the nucleotide analogs synthesized.

Figure 3

Figure 3

(A) The domain structure of the kinesin dimer. Kinesin residues including the head, neck, and a portion of the coiled-coil regions were expressed in bacteria and purified. (B) Coomasie-stained gels of the wild-type and mutant kinesins used in our assays. As assessed by gel filtration chromatography, these proteins eluted as soluble dimers.

Figure 4

Figure 4

(A) Three frames from a time-lapse fluorescence microscopy video of a kinesin-driven microtubule-gliding assay are shown. The distance rhodamine-labeled microtubules move in fixed time intervals determines the velocity. The images were acquired with a 60×, 1.4 NA Nikon objective and a Princeton Instruments cooled charge-coupled device camera. Bar = 2 μm. (B) The microtubule-gliding velocity of the wild-type kinesin in the presence of different ATP analogs (1 mM) is shown. Modification of the nucleotide with a Cp group yields an ATP analog that is incapable of driving kinesin-dependent movement. (C) Cp-ATP does not inhibit wild-type kinesin motility in the presence of ATP. AMPPNP at 3.5 mM completely inhibits kinesin motility in the presence of 2 mM ATP. The nonhydrolyzable Cp-AMPPNP does not reduce ATP-dependent kinesin motility. (D) A comparison of the gliding velocities of the different mutant kinesins in the presence of 1 mM ATP.

Figure 5

Figure 5

Characterization of the R14A kinesin mutant. (A) Measurement of microtubule-gliding velocities in the presence of different nucleotide analogs reveals that the mutant kinesin can use Cp-ATP more efficiently than unmodified ATP. (B) R14A kinesin-driven gliding velocities depend on the Cp-ATP concentration, and the data fit well to the Michaelis Menten model [velocity = _V_max∗[Cp-ATP]/(_K_m + [Cp-ATP])]. (C) Inhibition of R14A kinesin-dependent microtubule gliding by Cp-AMPPNP, in the presence of 1 mM Cp-ATP, shows a deviation from competitive inhibition at higher concentrations (broken line) and concentrations greater than 1 mM Cp-AMPPNP completely inhibit the mutant motor. This aspect of kinesin inhibition by AMPPNP has been characterized previously (27).

Figure 6

Figure 6

The specificity of the activator and the inhibitor for the R14A kinesin mutant. The kinesin superfamily member, Eg5, shows no detectable microtubule gliding in the presence of 2 mM Cp-ATP. Cp-AMPPNP also is unable to inhibit the enzyme at concentrations that unmodified AMPPNP completely inhibits its activity.

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